GSTs represent a multigene family of proteins consisting mainly of .alpha., .mu., .theta., and .pi.-class isoforms and are responsible for the detoxification of a range of xenobiotics, mainly via conjugation to glutathione. Alpha GST (.alpha.GST) is the major hepatic form and is uniformly distributed throughout the liver. In contrast, PiGST (.pi.GST) is located mainly in the epithelial cells lining the bile ducts. A similar unique distribution also exists with respect to the renal situation where .alpha.GST is located in the proximal tubules and .pi.GST is confined to the distal tubule region of the nephron. It can therefore be concluded that measurement of either plasma or urinary GST levels will facilitate the monitoring of either the hepatic or renal status of an individual. Numerous authors have, indeed, used GST measurements to evaluate both liver and renal damage in individuals who have undergone organ transplant or who have suffered organ damage by virtue of exposure to either hepatic or renal toxins (see, for example, Trull, A. K. et al., Transplantation (1994) 58, 1345-1351).
Currently such measurements are most usually performed using radioimmunoassays (RIA). However, recently a number of microtitre plate-based enzyme immunoassays (EIAs) have become commercially available from Biotrin International Limited, Mount Merrion, County Dublin, Ireland, which allow measurement of .alpha. and .pi.GST levels in biological fluids. These EIAs are now gaining widespread acceptance among the scientific community despite some initial scepticism (c.f. Beckett, G. J. and Hayes, J. D. (Advances in Clinical Chemistry (1993) 30, p.325)). Thus, at present RIA represents the detection method of choice for many workers in the field. Both of these techniques have certain limitations insofar as they require both specialised equipment and highly trained and competent personnel to generate meaningful and accurate results. Such assays are relatively slow requiring on the average 2 hours or more from start to finish. Accordingly, a more rapid assay format for GSTs is required.
Methods for the detection of GST isoenzymes are tending to become increasingly complicated as evidenced by the use of Time-Resolved ImmunoFluorometric Assay (TR-IFMA). Tiainen, P et al. (Clin. Chem. (1996) 42 2, 334-335) compared enzyme immunoassay (EIA) and TR-IFMA for .alpha.GST. The EIA used was Hepkit (trade mark of Biotrin International, Dublin). It was found that the inter-assay precision was better in Hepkit than in TR-IFMA. Also TR-IFMA is stated to have been somewhat more laborious than Heptkit because more replicates (four) and more washing cycles were needed to complete the assay procedure. Thus, it is clear that current research in to the detection and quantitation of GST has focussed on the development of more elaborate and complicated detection systems such as TR-IFMA.
Individuals who have undergone organ transplant or who have suffered organ damage by virtue of having been exposed to hepatic or renal toxins must be carefully monitored. This is especially true for individuals who have undergone organ transplant and who undergo a long post-transplant period of therapy with immunosuppressive drugs, in particular cyclosporin. Once such individuals are discharged from hospital they must be continuously monitored, which at present necessitates frequent returns to the relevant hospital unit, so that the requisite GST and other assays can be carried out. Accordingly, it would be most desirable if such persons had a means of self-monitoring which would reduce the number of hospital visits but, more importantly, would give an early indication to the patient and physician that intervention is required, in the event that an above-normal level of a GST is noted.
Thus, a method which would allow a faster and simpler format for GST detection in plasma, urine and other biological fluids, such as bile, would be a distinct advantage as it would allow the physician to monitor either the hepatic or renal status of an affected individual without recourse to time-consuming or expensive hospital-based tests.
Another area where a rapid assay format is required is in assessing organs prior to transplantation or in ex vivo experimentation. Currently, there is no generally acceptable method of donor organ evaluation prior to transplant into the recipient. Consequently it is impossible to accurately predict the final outcome of the transplantation operation as judged by the initial quality of the donor organ. Any method which would contribute to the pre-transplant assessment of the donor organ would greatly aid in the evaluation of the final outcome of the operation and may allow the physician to intervene earlier with post-transplant support therapy to prevent graft loss due to poor quality or host rejection.
As part of normal organ retrieval procedures, the donor organ is equilibrated or perfused with specific reagents (e.g. University of Wisconsin Buffer) once removed from the organ donor. This and other such reagents serve to maintain ex vivo organ integrity at low temperatures (4-10.degree. C.) prior to insertion into the recipient and are also used to equilibrate the stored organ to body temperature (37.degree. C.) prior to surgical reattachment to the recipient. Clearly, the quality of the donor organ will be dependent on many factors such as: donor health; time and temperature of ex vivo storage; handling procedures; and the quality of preservation reagents used. Thus, ex vivo graft injury, as caused by any of the above factors, could be monitored by evaluation of enzyme leakage from the organ.
Rapid assay formats are known. For example, EP 0 291 194 B1 describes and claims an analytical test device and an analytical method using said device which are suitable for use in the home, clinic or doctor's surgery and which are indicated to give an analytical result which is rapid and which requires the minimum degree of skill and involvement from the user. The device is indicated to be particularly suitable as a pregnancy testing device. No specific mention is made of GSTs. The levels of marker hormones in pregnant women, such as human chorionic gonadotropin (HCG) tend to be present in higher concentrations, and thus more readily detected, than the levels of GSTs in patients of the type hereinabove described and for whom elevated levels of GST are critical or, indeed, indicative of potential life threatening clinical events.
The difficulty of using enzyme activity measurements for discrimination between the isoforms of GSTs is well documented (Beckett G. J. and Hayes, J. D. (1993) supra). As indicated above, other investigators have used RIA (Howie, A. F. et al. (Clinica Chimica Acta (1989) 184, 269-278) and TR-IFMA (Tiainen, P and Karhi, K. K. Clin. Chem. (1994) 40-2, 184-189) to detect GSTs. Each of these techniques is a very sensitive technique for the detection of biomolecules. However, each technique requires specialised equipment and skilled personnel. For example, TR-IFMA requires the use of a special time-resolved fluorometer.
It will be appreciated that a technique is required which is suitable for non-experts or in a crisis situation which is rapid and easy to use. Representative of crisis situations would be liver damage or kidney damage post-transplant or potential liver failure due to paracetamol overdose.